The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster

Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot (esg)-GAL4 driver, expressing the yeast transcription factor gene, GAL4, under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of genes of interest in proximity to a GAL4-binding element, Upstream Activating Sequence, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, recent studies have shown that esg-GAL4 is also active in other tissues, such as neurons and ovaries. Therefore, the ISC/EB specificity of esg-GAL4 is questionable. In this study, we reveal esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene, RasV12, esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that esg-GAL4-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a “specific” ISC/EB driver to examine ISC/EB-mediated animal physiology.

overexpression of genes and various constructs to study fundamental roles of ISCs and EBs.For example, researchers have heavily used esg-GAL4 to generate ISC tumors by overexpressing oncogenic genes such as the gain-of-function transgenes, Ras and yokie (yki).esg-GAL4-driven ISC tumor animals have advanced our understanding of tumor-dependent impairment of systemic physiology, such as cachexia and the bloating phenotype.The crucial assumption for interpreting results of these studies as a phenotype originating from ISCs and EBs is that esg-GAL4 manipulates gene expression only in these cells in adults.However, recent studies reported that esg-GAL4 is expressed at least in some brain neurons 4 , ovaries 5,6 , and Malpighian tubules 7 , but not in muscles or fat bodies 4 .Therefore, the ISC/EB-specificity of esg-GAL4 has been questioned.
In this study, we report that esg-GAL4 is also expressed in the insect endocrine organ, the corpus allatum (CA), which is essential for synthesizing insect juvenile hormone (JH).Our data show that esg-GAL4-driven gene

Esg-GAL4 is expressed in the endocrine corpus allatum
We conducted experiments using the esg-GAL4 driver combined with tubulin promoter-driven temperaturesensitive GAL80 (tubP-GAL80 ts ).Hereafter, esg-GAL4; tubP-GAL80 ts is designated "esg ts -GAL4" or "esg ts > ".This strain has widely been used for adult stage-specific gene manipulation in ISCs and EBs 5,[8][9][10] .In all experimental conditions in this study, we reared esg ts > flies at a permissive temperature (21 °C) during development, such that esg-GAL4 activity is suppressed by GAL80 right before eclosion.Then, after eclosion, we subjected these flies to a restrictive temperature (29 °C) to activate esg-GAL4 only in the adult stage.
We realized by chance that esg-GAL4 was active in the tissue located at the most anterior part of the thorax in both males and females (Fig. 1b).More precisely, esg ts -GAL4-positive tissue was observed between the brain and proventriculus (Fig. 1c).This tissue was co-immunostained with an antibody against juvenile hormone acid O-methyltransferase (JHAMT), the essential enzyme that synthesizes JH in the CA 11,12 .This result strongly indicates that esg ts -GAL4-positive tissue between the brain and proventriculus is the CA.We also confirmed that esg ts -GAL4 was expressed in the CA of both male and female adult flies (Fig. 1d).Moreover, esg ts -GAL4 was expressed in the ring gland, particularly in the CA of wandering 3rd-instar larvae, as well as of adults (Fig. 1e).These results suggest that esg ts -GAL4 labels the CA in both male and female larvae and adults.
To confirm whether the esg gene itself is expressed in the CA, we used the esg-knock-in-GFP (esg-GFP) line 13 .As with esg ts > GFP expression, esg-GFP was expressed not only in a certain cells in the midgut, which seem to be ISCs/EBs (Fig. 1f) 13 , but also in the CA of both adult males and females (Fig. 1g).Furthermore, esg-GFP was expressed in the CA of wandering 3rd-instar larvae, while considerable expression of esg-GFP was also detected in other ring gland cells (Fig. 1h).These results suggest that esg is endogenously expressed in the CA.

RNAi of JH-biosynthetic enzyme by esg-GAL4 also impairs oogenesis
Next, we explored the possibility that esg ts -GAL4-driven transgenic RNAi suppresses gene expression in the CA.To examine this point, we conducted an RNAi experiment with esg ts -GAL4 to target jhamt, which is expressed explicitly in the CA 12 .Immunostaining signals of anti-JHAMT antibody were drastically decreased by jhamt RNAi, compared to controls (Fig. 2a,b; Supplementary Table S1).
In many insects, including D. melanogaster, JH promotes ovarian development by accumulating yolk components such as yolk protein and vitellogenin 14,15 .In D. melanogaster, a previous study reported that loss of jhamt activity results in smaller ovaries and reduced egg numbers 16 .Therefore, we observed ovary morphology and counted numbers of mature eggs in adult females expressing esg ts > jhamt RNAi.We found that esg ts > jhamt RNAi flies had smaller ovaries than controls (Fig. 2c).Consistent with this observation, the number of mature eggs was significantly decreased in RNAi flies (Fig. 2d; Supplementary Table S1).These results suggest that esg ts -GAL4-driven RNAi suppresses gene expression in the CA and influences JH-mediated biological events such as oogenesis.

Oncogenic Ras V12 expression by esg-GAL4 causes CA hypertrophy and abnormal expression of JH-responsive genes
In some recent studies, esg ts -GAL4 and UAS-Ras V12 have been utilized to induce ISC/EB tumors to investigate cell turnover in the midgut and tumor-mediated systemic physiology 8,17,18 .Since esg-GAL4 is also expressed in the CA, esg ts > Ras V12 might affect both ISC/EB and CA cells.Notably, esg ts > Ras V12 resulted not only in abnormal expansion of the esg ts -GAL4-driven GFP-positive area in the midgut (Fig. 3a) 17 , but also increased CA size and cell number (Fig. 3b-d; Supplementary Table S2).
Considering morphological abnormalities in the CA, it seemed possible that esg ts > Ras V12 expression enhances JH biosynthesis in the CA.Therefore, we next performed quantitative PCR on three JH-responsive genes, Krüppel-homolog 1 (Kr-h1), Jonah 25Bii (Jon25Bii), Odorant-binding protein 99b (Obp99b), to estimate the amount Figure 1.The transcription factor encoding gene, esg, was expressed in the CA.(a) (Left) GFP (green) driven by esg-GAL4 with tubP-GAL80 ts (esg ts >) was expressed in a subpopulation of adult midgut cells.(Right) magnified view of the area enclosed by the white square in the left figure.Blue is the DAPI signal.(b) esg > GFP, mCD8::GFP expression in whole bodies of adult males and females.Arrowheads and arrows indicate the CA and midguts, respectively.(c) esg ts > GFP (green) was expressed not only in some midgut cells, but also in the CA (arrowhead).This sample was derived from a female.The CA was labeled with anti-JHAMT antibody (magenta).Two inset images correspond to a region marked with a dashed line surrounding the brain, CA, and proventriculus.Both esg ts > GFP and anti-JHAMT immunoreactive signals were observed in the CA.(d) GFP (green) driven by esg-GAL4 labeled the CA in both adult males (upper) and females (lower).The CA was labeled with anti-JHAMT antibody (magenta).(e) GFP (green) driven by esg-GAL4 labeled the CA and a part of the prothoracic gland (PG) in wandering 3rd-instar (WL3) larva.The CA was labeled with anti-JHAMT antibody (magenta) and the PG was labeled with anti-Shroud (Sro) antibody (blue).(f) (Left) esg-knock in-GFP (green) was expressed in a subpopulation of adult midgut cells.(Right) A magnified view of the area encircled with a white line in the left figure.Blue is the DAPI signal.(g) esg-knock in-GFP (esg-GFP, green) was expressed in the CA in both males (upper) and females (lower).The CA was labeled with anti-JHAMT antibody (magenta).(h) esg-knock in-GFP (green) was expressed in the CA in WL3 larvae.The CA was labeled with anti-JHAMT antibody (magenta) and the PG was labeled with anti-Sro antibody (blue). of JH in Ras V12 overexpressors and controls 19 .Previous studies have shown that expression levels of Kr-h1 and Jon25Bii correlate positively with the amount of JH in the body, while Obp99b correlates negatively 19,20 .Our qPCR results showed that expression levels of Kr-h1 and Jon25Bii were increased by Ras V12 , while Obp99b was decreased (Fig. 3e-g; Supplementary Table S2).These results strongly suggest that esg ts > Ras V12 leads to abnormalities in CA cells and increased JH biosynthesis.

The CA-driver Aug21-GAL4 is not active in ISCs/EBs
A previous study has suggested that JH secreted from the CA is received by ISCs and/or EBs, enhancing ISC proliferation and leading to post-mating gut remodeling 5 .To overexpress genes to inhibit JH biosynthesis, the previous study utilized Aug21-GAL4, which is one of the most widely used CA-GAL4 drivers 21 .Since esg-GAL4 has high activity not only in ISCs/EBs, but also in the CA, we also verified whether Aug21-GAL4 is active in ISCs/EBs, as well as in the CA.We found that Aug21-GAL4-driven GFP signals, as well as anti-JHAMT immunoreactive signals, were present in the CA, but not in ISCs, EBs, or other types of midgut cells (Fig. 3h).This observation suggests that Aug21-GAL4-mediated manipulation of gene expression does not directly affect gene expression in ISCs and EBs.

Expression of other ISC/EB drivers in the CA
Lastly, we examined whether other GAL4 and LexA drivers used for gene manipulation in ISCs/EBs are also active in the CA.Beside esg-GAL4, many previous studies have used esg-LexA as an alternate binary gene expression driver active in ISCs/EBs [22][23][24][25] .We tested two esg-LexA drivers available from a stock center and found that both esg-LexA drivers were active in ISCs/EBs, but not in the adult or larval CA (Fig. 4a,b).It has also been reported that Delta-GAL4 and Su(H)-GAL4 are active in ISCs and EBs, respectively 26 .We found that the GFP signals driven by these GAL4 drivers were not present in the CA (Fig. 4c,d).These results suggest that the phenotypes resulting from genetic manipulation using these drivers are unlikely to be due to JH actions.Another recent study reported new genetic tools, intestinal-kickout (I-KCKT)-GAL4 and ISC-KCKT ts -GAL4, which are based in an intersectional method that restricts GAL4 expression to ISCs and/or EBs 27 (Fig. 4e,f).The study has confirmed that I-KCKT-GAL4 and ISC-KCKT ts -GAL4 are active in ISCs/EBs more specifically than conventional ISC/EB GAL4 drivers, such as esg-GAL4, Delta-GAL4, and Su(H)-GAL4, all of which are active in many types of cells other than ISCs/EBs 4,27 .We found that neither I-KCKT-GAL4 nor ISC-KCKT ts -GAL4 labeled the CA, suggesting that these KCKT-based GAL4 lines do not affect CA gene expression.

Discussion
In this study, we found that esg-GAL4, which is widely used to label midgut ISC/EB 2 , was also expressed in the CA.esg ts -GAL4 manipulates and influences gene expression in the CA, as esg ts -GAL4 > jhamt RNAi decreases mature egg formation, which is a typical phenotype of reduced JH titer.Whereas esg-GAL4 is active in some ovarian cells 5,6 , it is reported by FlyBase (https:// flyba se.org/ repor ts/ FBgn0 028841.html) that jhamt expression is not detected in the ovary.Therefore, the mature egg number phenotype by esg ts -GAL4 > jhamt RNAi is due to the knock-down of jhamt expression in the CA but not the ovary.Moreover, esg ts > Ras V12 caused CA hypertrophy and influenced JH-responsive gene expression (Fig. 3b-g), suggesting that esg ts > Ras V12 increases JH biosynthesis.Our data suggest that esg itself is expressed in the CA in both males and females of both larvae and adults.Enrichment of esg expression in the larval ring gland and the adult CA were suggested by a previous microarray analysis 28 and a recent single-cell RNA-seq analysis 29 , respectively.esg encodes a Snail-type transcription factor that contributes to cell cycle regulation, cell differentiation, and cell-cell adhesion in many cell types in D. melanogaster [30][31][32][33] .However, functions of Esg that regulate differentiation and morphogenesis of the CA have not been studied.Thus, additional studies are needed to clarify how Esg is involved in CA cell regulation, especially whether it regulates JH biosynthesis.
In D. melanogaster, one of the reported functions of JH is that this hormone directly acts on ISCs and EBs through nuclear JH receptors, Methoprene-tolerant (Met) and Germ cell expressed (Gce), to regulate gut remodeling in mated or aged females 5,8 .Interestingly, previous studies reported that esg ts > jhamt RNAi reduces numbers of ISCs and EBs.This phenotype is also observed in esg ts > Met or gce RNAi animals.Since these studies use esg ts -GAL4 as the ISC/EB-"specific" GAL4 driver, these papers propose that JH is biosynthesized in ISCs and EBs outside the CA, and cell-autonomously regulates maintenance of ISCs and EBs during aging 8 .However, our data strongly indicate that esg-GAL4 is also expressed in the CA.More importantly, esg ts > jhamt RNAi causes a decrease in JHAMT protein in the CA (Fig. 2a), which implies decreased JH biosynthesis in the CA, hence the systemic decrease in JH titer.We emphasize that although these previous studies did not examine jhamt expression in ISCs and EBs, they carefully evaluated functions of JHAMT in ISCs and EBs with additional experiments in which they utilize other GAL4 drivers to knock down jhamt in ISCs and EBs via Delta-GAL4 and Su(H)-GAL4, respectively 8 .Therefore, we argue that JH biosynthesis most likely occurs in ISCs and EBs, although we cannot detect obvious anti-JHAMT reactivities in the gut region (Fig. 1c, 3h).
Nevertheless, a previous study using Aug21-GAL4, the well-known CA-GAL4 driver 21 , revealed that Aug21-GAL4-mediated inhibition of JH biosynthesis in the CA suppresses ISC proliferation 5 .Aug21-GAL4 is not active in ISCs/EBs (Fig. 3h), suggesting that the Aug21-GAL4-driven ISC phenotype cannot be explained by JH biosynthesis in ISCs/EBs.Taken together, we cannot rule out the possibility that JH is also supplied from the CA for maintenance of ISCs and EBs.In addition, ISC/EB increased by JH derived from the CA may further enhance JH production in the gut, implying that CA-ISC/EB interactions are more complex than previously thought.In the future, it will be important to investigate how much CA and ISC/EB each contribute to the circulating JH levels.
In the last decade, esg-GAL4 has widely been used to generate ISC tumors by overexpressing oncogenic genes such as the gain-of-function transgenes, Ras, Raf, and yki 17,34 .In particular, very recently, a number of studies have utilized esg-GAL4-driven oncogenic gene models to study how ISC tumors impact gut homeostasis, as well as systemic physiology 35 .However, based on our results, when interpreting these esg-GAL4-driven phenotypes, we should consider not only effects of ISC/EB tumors, but also effects of JH biosynthesis abnormalities caused by CA hypertrophy.For example, some intestinal cells receive JH from the CA through Met and Gce, influencing gut remodeling 5,36 .Beside Ras V12 , recent studies have shown that esg ts > yki 3SA leads to severe cachexia and bloating phenotypes, mediated by abnormal hormone secretion from organs such as Malpighian tubules and midgut 9,23,37,38 .Considering the systemic nature of these ISC/EB tumor-associated phenotypes, it may be necessary to consider the function of JH, which has major impacts on insect physiology.Based on observations in this and a previous study 27 , I-KCKT-GAL4 and ISC-KCKT ts -GAL4 seem to be the current best choice of GAL4 drivers to manipulate gene expression specifically in ISCs and EBs without influencing the CA.However, even I-KCKT-GAL4 is active in Malpighian tubules 27 .Generally speaking, it will be important to examine phenotypes using more than just one GAL4 driver.

Figure 2 .Figure 3 .
Figure 2. jhamt-RNAi driven by esg-GAL4 inhibited jhamt in the CA and reduced the number of eggs in the ovary.(a) Immunostaining signal of anti-JHAMT (magenta) in the CA was drastically decreased by jhamt-RNAi, driven by esg-GAL4.Upper panels are controls (esg ts > GFP) and lower panels are jhamt-RNAi (esg ts > GFP, jhamt-IR KK ) flies.The CA is encircled with a dashed line.(b) Quantification of anti-JHAMT antibody immunostaining signals normalized by the GFP signal between control and jhamt-RNAi flies.(c) Ovaries of control (upper: esg ts > GFP) and jhamt-RNAi (lower: esg ts > GFP, jhamt-IR KK ) virgin females.(d) Numbers of mature eggs in virgin females were significantly decreased by adult-specific jhamt-RNAi driven by esg-GAL4.For each experimental condition, 20 adult females were used.The Wilcoxon rank sum test was used for these data.*P < 0.05, **P < 0.01, ***P < 0.001.